1. Field of the Invention
The present invention relates to a reagent and a method for detecting an antigen, antibody or other analyte in human or animal blood by erythrocyte agglutination. The invention also concerns a kit containing the reagent and processes of preparation of the reagents.
2. Information Disclosure Statement
Assaying blood samples for a particular antigen or antibody has traditionally involved the step of separating the cellular components from the serum components of the blood by centrifugation and/or clotting, prior to assay.
This presents several potential problems. Firstly, such an assay is not suited to testing being conducted under field conditions. In many veterinary situations a quick test in the field is more desirable than the alternative of transporting samples to laboratories for separation and assay. Also, veterinary surgeons who do not have access to a centrifuge frequently need to assay blood samples for the presence of infectious agents such as heartworm. Further, assays being used for the detection of diseases in Third World countries present a situation where simplicity and low cost are of the essence.
Secondly, in certain pathologic conditions, separation of the blood samples becomes difficult. Blood taken from patients suffering conditions such as Waldenstrom's macroglobulinemia is difficult to separate into serum and cell fractions making an assay which can be conducted on whole blood highly desirable.
Thirdly, blood samples are often used for testing for the presence of highly contagious and potentially dangerous disease states. In these cases it is preferable that as little handling and processing of the samples as possible is undertaken in order to minimize the risk to personnel conducting the assay. Further, certain conditions make the provision of over-the-counter finger-prick assays highly desirable. Such assays must necessarily be suited to performance on whole blood.
Immunoassays have revolutionized human diagnostic and veterinary medicine since the introduction of techniques such as the radioimmunoassay, first reported by Yalow and Berson (1959) Nature 184, 1648, and the enzyme immunoassay or EIA which was first reported by Engvall and Perlman (1971) Immunochem 8, 871 and Van Weeman and Schuurs (1971) FEBS Letters 15, 232.
Whilst such assays are based on antibody-antigen interactions the detection systems utilized are usually complex. The reagents used are generally enzyme or radiolabelled antigens, antibodies or complexes thereof which require either incubation with specific substrates and measurement of a color end-point either visually or by means of a colorimeter or measurement of radioactive decay with radiation counters to detect the presence of the analyte being tested. These assays also involve several washing steps. Most immunoassays for the detection of analytes in blood are currently of this nature. Thus, whilst these assays are sensitive, they are lengthy and involved procedures which may require expensive instrumentation, for detection of the analyte under test.
An alternative to these assays is provided by immunoassays of the type described by Gupta, et al., (1985) Journal of Immunological Methods 80:177-187. These are immunoassays in which erythrocytes and anti-erythrocyte antibodies are used in the indicator system. In these assays exogenous erythrocytes such as sheep erythrocytes are used.
In recent years it has been possible to attach antibodies to latex beads, thus providing a rapid agglutination assay. This, however, still entails the separation of the serum/plasma phase from the cellular phase and consequently requires the use of a centrifuge or filtration system. Latex agglutination assays are described in Castelan, et al., J. Clin. Pathol. (1968), 21, 638; and Singer & Plotz Am. J. Med. [1956 (Dec)], 888.
Both direct and indirect agglutination immunoassays are well known in the art. In these assays, the agglutination of particles to which antigen or antibody is bound is used to indicate the presence or absence of the corresponding antibody or antigen. A variety of particles, including particles of latex, charcoal, kaolinite, or bentonite, as well as both microbial and red blood cells, have been used as agglutinatable carriers. See Mochida, U.S. Pat. No. 4,308,026. The use of erythrocytes as indicator particles is strongly criticized by Patel, U.S. Pat. No. 3,882,225, who says that it is difficult to standardize indicator erythrocytes.
Molinaro, U.S. Pat. No. 4,130,634 describes an assay for an antigen which employs antibody-coated red blood cells. Molinaro emphasizes that the method used to couple the antibody to the erythrocyte must not destroy the reactivity of the antibody. He makes it clear that antibodies which are specific for the erythrocyte are not useful for his assay. He does mention, however, the possibility of using a hybrid antibody with one binding site specific for the antigen and the other specific for the red blood cell.
Chang, U.S. Pat. No. 4,433,059 discloses an agglutination immunoassay reagent in which two antibodies are covalently linked "tail-to-tail", i.e., so as not to alter their specificity. One antibody is specific for an antigen borne by an indicator substance, such as an erythrocyte. This antibody is preferably univalent to avoid nonspecific agglutination. The other antibody is divalent and is specific for the analyte. In preparation for the assay, fresh erythrocytes are coated with the conjugate. The double antibody conjugate-coated RBCs are then incubated with the test serum. Chang does not contemplate the assaying of whole blood samples using a non-autoagglutinating anti-RBC antibody and endogenous erythrocytes. He does not recognize that nonspecific agglutination may be avoided without resort to univalent erythrocyte binding fragments or address the use of binding substances other than antibodies. Additionally, he teaches that the analyte and RBC binding antibodies should be coupled by means of a heterobifunctional coupling agent.
Cytosignet, Inc., W088/05913 (published 11 Aug. 1988) broadly describes an erythrocyte agglutination assay which detects an analyte in an erythrocyte-containing sample by means of a conjugate of an analyte-binding moiety and an erythrocyte-binding moiety (especially an antibody or lectin). While Cytosignet recognizes the need for constructs "which, in the absence of analyte, react only with particulates in the sample in such a manner that detectable agglutination of particulates does not occur," it is oblivious to the possibility of using an intact antibody, such as the anti-glycophorin antibody discussed later herein, as the erythrocyte binding element. Instead, it teaches use of an univalent Fab fragment of an anti-RBC antibody, which, like that of Chang, is derived from a divalent antibody which is capable of cross-linking two erythrocytes and thus of forming an agglutinate. Nor does it teach how to prepare a lectin so that "auto-agglutination" is avoided. Finally, it does not delineate any binding molecules, other than antibodies or lectins, which are useful in the proposed assay.
Smith, U.S. Pat. No. 4,578,360 and U.S. Pat. No. 4,401,764 describe a conjugate of an erythrocyte binding molecule and a label binding molecule. His label is a fluorescent, chemiluminescent, spin, enzyme or coenzyme label; an agglutination assay is not contemplated.
Smith, W088/05913, bearing an international publication date of 11 Aug. 1988 but claiming priority from U.S. Ser. No. 8,571 of 29 Jan. 1987, describes an agglutination assay for an analyte in a whole blood sample which uses a construct comprising a RBC-binding moiety and an analyte-binding moiety, coupled by means of a bifunctional coupling agent. The exemplified RBC-binding moiety was a Fab' (i.e., univalent) fragment of a RBC-agglutinating antibody. The use of an anti-glycophrin monoclonal antibody (or a divalent binding fragment thereof) or of a mellitin fragment as the particulate-binding moiety is not taught.
Chu, U.S. Pat. No. 4,493,793 discloses the construction of a lectin-antibody or lectin-antigen covalently coupled conjugate. His Table I (incorporated by reference) sets forth the carbohydrate specificities of several lectins. He does not teach coupling such a conjugate to an erythrocyte through either the lectin or the antibody receptor.
Other "tail-to-tail" immunological conjugates are known. Segal, U.S. Pat. No. 4,676,980 sets forth the construction of a "tail-to-tail" conjugate of a target cell surface antigen-specific antibody and of a cytotoxic effector cell receptor-specific antibody. Several cross-linking methods, incorporated by reference, are described. This conjugate is intended for use in immunotherapy, in that it will cause the cellular immune system of the patient to lyse the target cell. The target cell would not, of course, be an erythrocyte endogenous to the host.
Freytag, U.S. Pat. No. 4,517,303 describes an immunolytic assay employing a conjuqate of an analyte molecule and a cytolysin. The cytolysin may be (whole) mellitin. Since the Freytag assay requires that the conjugate lyse red blood cells, use of mellitin 7-26 or other nonlytic, RBC-binding peptides would be contrary to Freytag's teachings.
Li, U.S. Pat. No. 4,661,444 suggests the production of a tail-to-tail conjugate of an analyte-binding antibody and of an antibody specific for the idiotype of the first antibody. This conjugate was to be used in conjunction with an insolubilized analyte-binding antibody in an immunoassay.
Wardlaw, U.S. Pat. No. 4,695,553 teaches use of a monoclonal antibody against a universal erythrocyte antigen as a RBC agglutinating agent to clarify the interface between red blood cells and white blood cells in centrifuged whole blood. He prefers use of antibodies against glycophorin or against H antigen, but also mentions the possibility of using a mixture of lectins. Guesdon, U.S. Pat. No. 4,668,637 discusses the use of anti-red blood cell antibodies or of lectins for the purpose of erythroadsorption. Bigbee, Molecular Immunology, 20: 1353-1362 (1983) describes the production and testing of four monoclonal antibodies against glycophorin A. The general concept of using in an immunoassay an antibody which reacts with an antigenic determinant shared among all members of a class of analytes of interest (microorganisms) is set forth in McLaughlin, U.S. Pat. No. 4,683,196.
A number of patents deal with antibodies useful in blood typing. See, e.g., Lloyd, U.S. Pat. No. 4,678,747; Graham, Jr., U.S. Pat. No. 4,358,436; Liu, U.S. Pat. No. 4,550,017; Steplewski, U.S. Pat. No. 4,607,009; Lennox, W083/03477. These antibodies are useful for blood typing because they bind to antigens found only in certain blood cell populations, while for the purpose of this invention, it is desirable to use antibodies (or mixtures thereof) which bind to essentially all erythrocytes.
Zuk, U.S. Pat. No. 4,594,327 recognizes the desirability of performing an immunoassay directly on whole blood samples. In his method, the sample is contacted with both an insolubilized, analyte-specific immunoreagent and with a red blood cell binding agent such as a RBC-specific antibody or a lectin. The analyte-specific immunoreagent and the RBC binding agent are not coupled together, and the assay disclosed is not an agglutination assay. Rather he provides assay means which filter out the red blood cells and then expose the remainder of the sample to the immunoreagent.
The problem, in an agglutination immunoassay, of nonspecific agglutination of erythrocytes by anti-erythrocyte antibodies endogenous to the blood sample, was noted by Czismas, U.S. Pat. No. 3,639,558. He proposed eliminating all naturally occurring antigenic sites on the particle by coating the particle with protein.
Theofilopoulos, U.S. Pat. No. 4,342,566; Duermeyer, U.S. Pat. No. 4,292,403 and Goldenberg, U.S. Pat. No. 4,331,647 are of interest as demonstrating the use of specific binding fragments of antibodies as substitutes for intact antibodies in assays. The construction of heterobifunctional antibodies is taught by Auditore-Hargreaves, U.S. Pat. No. 4,446,233; Paulus, U.S. Pat. No. 4,444,878; and Reading, U.S. Pat. No. 4,474,893. Mochida, U.S. Pat. No. 4,200,436 discloses the use of monovalent antibodies or binding fragments thereof in certain immunoassays. Forrest, U.S. Pat. No. 4,659,678 mentions that monovalent antibodies cannot form dimers or more extensive complexes with the antigen; such aggregates were said to be capable of interfering with the binding of the antigen-antibody complex to a solid phase support.